Fluorescence detector, and a device for supporting a replacable sample cuvette in a fluorescence detector

ABSTRACT

A fluorescence detector has a light source in the form of a halogen lamp, a UV-filter which allows excitation radiation in UV to pass through, and a second filter which sorts out fluorescence radiation, and a detector which detects the through-passing fluorescence radiation. The filters comprise thin straight blocks and also function as light guides and are positioned at right angles with their extensions directed towards a point at which a tubular sample can be placed in close with the ends of the filters distal from the light source and the detector.

FIELD OF THE INVENTION

The present invention relates to a fluorescence detector for measuringfluorescence in a liquid contained in a tube, comprising a light sourcefor irradiating the liquid with fluorescence-generating radiation, afilter through which fluorescence radiation emitted by the liquidpasses, and a detector which functions to detect fluorescence radiationthat has passed through the filter and to produce an electric signalcorresponding to said fluorescence radiation.

The invention also relates to a carrier device for a cuvette intendedfor simple, reproducible replacement in a fluorescence detector.

BACKGROUND OF THE INVENTION

Many different makes of apparatus for activating and detectingfluorescence radiation from liquids are commercially available. Theliquid is introduced into a cuvette or is allowed to flow past ameasuring point, where it is irradiated with UV-light from a lightsource, while resultant fluorescence radiation is sorted out and passedto an intensity measuring detector. In many instances monochromators areused both for producing radiation and for sorting out fluorescenceradiation of a given wave-length. Although it is possible to manufacturesuch instruments with a high degree of sensitivity and therewith allowThe detection of small concentrations, such instruments are unavoidablyvery expensive. Simpler instruments which operate solely with colourfilters are also available, although these instruments are not able tosatisfy the same high requirements at present.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a fluorescence detectorwhich is simple and inexpensive in manufacture but which, nevertheless,will provide high detection sensitivity. The detector is primarilyintended to be used with through-flowing samples from smallchromatography columns or in so-called flow injection systems, althoughit can also be used with static samples. In the case of the applicationsfor which the invention is primarily intended, it is possible to userelatively broad wavelength bands for both the incident excitingradiation and the captured emitted radiation. This is possible becauseof the high degree of sensitivity that can be obtained due to theeffective use of the light, and because it is possible to use colourfilters in the majority of cases, provided that these filters aresuitably constructed.

Another object of the invention is to provide a simple and practicalexchange possibility for cuvettes and samples mounted in exchangeablecassettes.

These and other objects of the invention are achieved in accordance withthe invention with a fluorescence detector having the characteristicfeatures set forth in the claims.

One particularly distinguishing feature of one embodiment of theinvention is that the filters used have the form of plates cut fromfilter material and fulfill two different functions simultaneously,namely a filter function and a light conducting function. In this way,there is achieved both a good filter effect for long wavelengths throughthe filters and favourable collimation, despite the fact that no lensesare required. However, the filter effect can also be achieved by placingseparate filters in the beam paths.

With regard to the carrier device or the cassette according to theinvention, one characteristic feature resides in the use of an innercomer in the detector, in the form of a surface from which fluorescenceexciting light is emitted towards a cuvette or the like, and a surfacewhich is angled in relation to the first-mentioned surface and throughwhich fluorescence light is taken up for detection, wherein the cuvetteor like device is mounted resiliently in the cassette and is pressed intowards the two surfaces to take a fully reproducible position whenmounting the cassette.

Similar to known fluorescence detectors, there is used a sample cuvettewhich consists in a thin tube of transparent material, preferablyquartz, through which the examined liquid flows. The quartz tube ismounted advantageously so as to abut the short sides of the two filters.Particularly when the tube and associated connections are mounted in areplaceable or exchangeable cassette, an advantage is obtained when thecassette includes resilient means which, when the cassette is fitted,resiliently press the outer wall surface of the tube against the shortsides of the tube filters, said filters preferably being mounted in aholder having an inner corner angled at 90 degrees.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail with reference tonon-limiting exemplifying embodiments thereof and also with reference tothe accompanying drawings, in which

FIG. 1 is a sectioned view of a fluorescence detector constructed inaccordance with the principles of the invention, with no sample mounted;

FIG. 2 is an enlarged view of a sample position and shows a quartz tubein cross-section and a radiation delivering and fluorescence radiationcollecting optical element to those parts that are close to the sample;

FIG. 3 illustrates a cassette having a resiliently mounted quartzsample-tube;

FIG. 4 is a longitudinal sectioned view of a quartz sample tube;

FIG. 5 illustrates an electric circuit of a photodiode withpreamplifier; and

FIG. 6 illustrates a variant of the embodiment shown in FIG. 1 in whicha normalizing device of the two-beam type is arranged.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates a fluorescence detector in accordance with oneexemplifying embodiment, the detector being shown in twice its naturalsize. Mounted in a holder 1 of solid black plastic at right angles toeach other are a first light conductor 2 which also functions as afilter which is permeable to ultra-violet light, and a second lightconductor 3 which also functions as a filter which is permeable tofluorescence light, wherein the two light conductors meet at theirrespective one ends to form an inner comer. A bulb 4 is mounted at thedistal short end of the first light conductor, while a detector unit 7is seated on the distal short end of the second light conductor. Thesetwo filters, which have the form of a right-angled block, are rigidlyattached in the holder 1, with light-impervious, black thermosettingresin 6 (preferably silicone rubber) surrounding said meeting ends, sothat only the their short-side surfaces are exposed. The end of thelight conductor/filter 2 that lies proximal to the bulb 4 projectsslightly out from the holder 1 and a portion of said projecting partextends into the bulb housing. The bulb 4 is mounted in a lamp housing(not shown) and is there ventilated by a fan, which also functions tocool that part of the filter which protrudes from the holder 1. As aresult, only a small amount of heat is conducted to the sample. Areflector 5 may optionally be placed behind the bulb 4. The detector 7will preferably be a photodiode whose sensitive surface is placedtowards the short side of the light conductor 3 and an intermediateplastic filter is provided to eliminate any transmitted UV-radiationthat may have successfully passed through the filter effect of the lightconductor 3.

FIG. 2 illustrates schematically the corner formed by the two lightguides 2 and 3, this view being enlarged 35 times, and a quartz tube 10fitted therein through which or in which a liquid sample can be passedor stored respectively. Fluorescence-generating excitation radiationarrives in the direction of the arrow towards the tube and generatestherein fluorescence radiation, provided that the liquid contained inthe tube fluoresces. Part of this fluorescence exits from the tube andenters the light guide 3, where it is filtered to eliminate anyUV-radiation that may have been diverted in this direction, bydispersion, reflection or refraction. The actual tube 10 will preferablybe free from fluorescence, and consequently the tube is normally made ofquartz.

In order to increase intensify, the tube 10 may be provided partiallywith a reflective coating on its outer surface, preferably an aluminumcoating. FIG. 2 illustrates the preferred application of the reflectivecoatings 20 and 21, each covering 76 degrees with an interspace 22 of 40degrees. It will be understood that these values are proposed values andthus not critical. It is important, however, that there is found anintermediate space in a field around the location of the bisector 23between the pupil surfaces of the light conductors 2 and 3, so as toprevent the excitation radiation being directed towards the outlet pupilto any great extent, via reflection.

The radiation geometry has therewith been shown in cross-section. Thiscross-section is representative of the geometry that prevails over agiven length of the tube 10, which considerably exceeds thecross-sectional dimensions shown in FIGS. 1 and 2. These illustratedcross-sections are sections through the thickness of the lightconductors/filters 2 and 3, which have the form of elongated plates withtheir largest extension and their smallest extension shown in FIG. 1.

It will be noted that although it is normally preferred to usecylindrical tubes, it is also possible to use cuvettes and tubes ofother shapes when this is practical for some reason or another. It isthus obvious that a tube of square cross-section can be fitted in thesame optical system without detriment.

The tube 10 is thus mounted at the point of intersection of the opticallight conductors 2 and 3, as shown in FIG. 2. According to thispreferred embodiment, the tube 10 is replaceably mounted in a cassette,such as via flexible and resilient steel pipes 11 in a cassette 12according to FIG. 3. The holder 1 shown in FIG. 1 is mounted togetherwith a cooled lamp housing in a casing in a manner not shown, saidcasing having an opening into which the cassette 12 can be slid, in themanner of a sliding box. The cassette 12 is secured by means of a screw14 after having been slid into the opening. The tube 10 then extendsobliquely from beneath towards the inner comer in FIG. 1 and is held inresilient contact by the flexible stainless pipes 11.

FIG. 4 illustrates how the tube 10 is sealingly connected to the steelpipes 11. In this regard, the pipes 11 are collared so as to provide aclose fit with the inner surface of the tube 10, and a pipe 41 made ofplastic-elastic material, for instance FEP, is fitted, followed by anapertured plug. The plug 42 pushes against the pipe 41 therewith causingthe pipe to deform and seal in the space between the inner surface ofthe tube 10 and the outer surface of the steel pipe 11, and is loadedwith a spring 43 tensioned with a further apertured plug 44 which isheld in place by the tension of the pressure spring 43, by virtue of abend in the pipe 11. As will be seen from FIG. 3, the pipes 11 are thencurved, so that the tube 10 is kept free between connections 13, whichpermit connection to a liquid source, for instance a chromatographiccolumn. When a quartz tube is used, it is convenient to provide on oneside opposite to the tube abutment side a reinforcing bar which joinsthe pipes 11 to the joins.

The light source used in the illustrated case is a halogen lamp 4 whichis mounted in a jig 50 comprising a plate 51 provided with a hole 52 forreproducible mounting. An electrical connector is provided withelectrical connections. In the described embodiment, there is used a12V, 20 W bulb (model Q20 G4) with the filament extending parallel withthe entry surface of the first filter 2 and with the axial direction ofthe tube 10.

It has been found that for normal requirements, it is sufficient toprovide a stabilized d.c. voltage for supplying the bulb with energy,i.e. so as to obtain good long term stability over a test period,including any calibration necessary. As will be made apparent below, itis also possible to monitor the intensity by means of a two-beam device.

The detector used is suitably a photodiode, which is beneficial in viewof the fact that it has good linearity over a large dynamic range (4-5powers of ten). As illustrated schematically in FIG. 5, thelight-quanta-generated current is passed through a high-resistiveresistor (10 Gohm) and the signal obtained is amplified in apreamplifier. The resistor and the amplifier are positioned in the closeproximity of the photodiode and are well screened. The amplified signalis preferably led to a voltage/frequency converter, this being suitableas the operations concerned are relatively slow. The diode used is aphotodiode. A Hamamatsu photodiode, model designation S 2387-16, wasused in one construction.

The combined light conductors and filters are suitably manufactured bycutting commercially available glass filter plates into strips, theseplates often being available in square shapes having a thickness of 1mm, for instance. A width of about 10 mm is appropriate, and the lengthwill preferably be much greater than the thickness, particularly in thecase of the UV-filter, which will preferably be at least 10 timeslonger, preferably at least 50 times longer than the thickness, whereasthe intake filter will be at least 6 times longer, preferably at least10 times longer than the thickness. The cut edge surfaces of the filterstrips cut from said plates are ground and polished, without anyparticular optical tolerance requirements. Suitable filters can have apass band, the UV-filter somewhere between 300 and 380 nm, the secondfilter between 400 and 500 nm, although special requirements can besatisfied by individual adaptation of the filters. The best length ofthe filters is determined in view of the type of filter used.

In one constructed example of a fluorescence detector, there was used aquartz tube which had an outer diameter of 1.5 mm and an inner diameterof 1.1 mm. The thickness of the filter glass used was 1 mm. TheUV-filter was made by Schott, type UG5 (pass band 310-350 nm) andmeasured 49×6×1 mm. The second filter, also a Schott make, type BG 39(pass band 410-480 nm), measured 12×6×1 mm. The end of the detector wassupplemented with a small plastic filter impervious to UV(non-transparent under 390 nm, type KV 389 from Schott).

Comparisons have been made with commercially available fluorescencedetectors. It was found that with an irradiated cell volume of 5.7 μl,the detection limit of the standard substance quinine sulphate was 0.010pg/μl (with the detection limit defined as a signal level reaching totwice the background noise). Sensitivity is therewith essentially on alevel with what, according to available data sheets, is said to havebeen achieved with the best of the tested instruments generallyavailable commercially.

As before mentioned, it has been found that the use of a single-beamsystem in accordance with what has already been described is generallysufficient for illumination. However, if particularly high requirementsare placed on the stability and reproduceability of the measuringprocess, there can be provided a reference system for measuring theradiation emitted from the bulb, for instance in accordance with FIG. 6,where like details have been identified with like reference signs. Therehas been drilled in the light conductor 2 a hole 60 which takes-up asmall part of its width (less than 10-15%). This hole has placedtherein, for instance by embedment, a plug of fluorescent material whichwhen impinged upon by the radiation led through the light conductoremits fluorescence radiation of which a part is captured by a lightconductor 3b and led to a reference photodiode 7b, which thus detects asignal that is a measure of the excitation radiation. This enables thefollowing advantages to be achieved:

Because part of the luminescence that passes towards the measuringcuvette is taken-out as a reference, the measured light and thereference light are both taken from the same part of the light sourceand in the same direction.

Because the reference beam has passed through a substantial length ofthe light conductor 2, the reference beam will have generally the samespectral composition as the excitation beam that impinges on the samplewhen the reference beam strikes the reference plug, provided that thelight conductor has a filtering effect. In addition, if the lightconductor 2 has a filtering effect, the fluorescence radiation generatedby the plug will be absorbed as a result of the position of the plugbefore the radiation can reach the sample, therewith scarcelyinfluencing the background.

The reference plug can be produced from a fluorescent material whosefluorescence properties are similar to those exhibited by samples to beanalyzed.

Because the photodiodes 7 and 7b have been placed close together andmechanically mounted in similar ways, several advantages are achieved,for instance the photodiodes can be connected electrically to the samecircuit board on which the two amplifiers are mounted, and the twophoto-diodes can be readily held at the same temperature.

The plug may be replaced with a mirror positioned at 45 degrees, a totalreflecting prism or some light-spreading device, to transmit part of theexcitation light through the light guide 3b. In this regard, theproperty according to the latter point is not relevant and in this case,it is necessary to produce the light guide 3b from a material which willallow the excitation light to pass through.

I claim:
 1. In a fluorescence detector for measuring fluorescence in aliquid comprising a light source for irradiating the liquid withfluorescence generating radiation, a filter for allowing fluorescenceradiation generated by liquid to pass through, and a detector fordetecting fluorescence radiation that has passed through the filter andfor producing an electric signal corresponding to the fluorescenceradiation, the improvement comprising:an elongated, flat-shaped firstlight conductor having a first end in proximity to the light source, anda second end adapted to be positioned in proximity to an irradiatedregion of a liquid-containing tube having an inner diameterapproximating the thickness of the first light conductor, said firstlight conductor having a length which is at least 10 times saidthickness, and functioning as a filter to provide a first filter effectwhich is pervious to ultraviolet light but impervious to wavelengthranges of generated fluorescence radiation; an elongated, flat-shapedsecond light conductor having a first short side in proximity to thedetector, and a second short side adapted to be positioned in proximityto a detection region of the tube whose inner diameter approximates thethickness of the second light conductor, said second light conductorhaving a length which is at least 6 times that of its thickness, saidsecond light conductor being pervious to fluorescence radiation andfunctioning as a filter to provide a second filter effect; wherein thelight conductors are positioned with their longitudinal axes in mutualtransverse directions, and wherein in use, the second end of the firstlight conductor and the second short side terminate almost tangentiallywith the outer surface of the tube, and the irradiated region and thedetection region coincide at least partially in the longitudinaldirection of the tube.
 2. A fluorescence detector according to claim 1,further comprising an exchangeable cassette for mounting the tubetherein.
 3. A fluorescence detector according to claim 1, wherein thelight source is a halogen bulb contained in a quartz casing.
 4. Afluorescence detector according to claim 1, wherein the detector is aphotodiode.
 5. A fluorescence detector according to claim 1, wherein theouter surface of the tube is coated partially with a reflective layer,which in use leaves exposed a surface of the tube that is located at aregion around a bisector between the two surfaces of the lightconductors that are closest to the tube.
 6. A fluorescence detectoraccording to claim 1, wherein the filter effects of the light conductorsare achieved by producing the light conductors from filter material. 7.A fluorescence detector according to claim 6, further comprising areference system which includes a fluorescent reference sample mountedin the first light conductor between the first end and second end, athird light conductor having one end directed towards the referencesample, and an intensity monitoring second detector arranged at anopposite end of said third light guide.
 8. A fluorescence detectoraccording to claim 1, further including means for pressing the tuberesiliently against the light guides.
 9. A fluorescence detectoraccording to claim 8, wherein metal inlet and outlet pipes havingcollared ends are inserted into both ends of the tube, followed bytubular members which are made of a deformable plastic material andfitted over the inserted metal pipes, and plug means which are made of aharder material and are press-fitted into the tube, said plug meansbeing resiliently pressed against the fitted tubular members.
 10. Afluorescence detector according to claim 1, wherein the first filtereffect has a pass band of between 310-350 nm, and the second filtereffect has a pass band between 410-480 nm.
 11. A fluorescence detectoraccording to claim 10, wherein the second filter effect is combined witha plastic UV-filter.
 12. A fluorescence detector according to claim 1,further comprising a cuvette in the fluorescence detector, means forfitting the carrier device and an attachment end thereof to thefluorescence detector in a specific mounting position, wherein thecuvette is suspended from the carrier device via resilient inlet andoutlet lines, so that after fitting the carrier device to thefluorescence detector, the cuvette is pressed by support surfacesprovided in the fluorescence detector from a first position to a secondmeasuring position which can be reproducibly well-defined in relation tothe fluorescence detector and its support surfaces.
 13. A fluorescencedetector according to claim 12, wherein the cuvette is made of quartzglass and is reinforcingly supported between the inlet and outlet linesby a bar which joins said lines on one side of the cuvette that liesdistal from the support surfaces of the fluorescence detector.